siRNA: a promising new approach to anti-influenza therapy
<The what and how of siRNAs>
siRNAs are 21�25-nucleotide RNAs that act posttranscriptionally to induce homologous sequence-dependent degradation of mRNAs. The native process of sequence-specific silencing involves long double-stranded RNAs (dsRNAs). Long dsRNAs are processed in the cell by the dsRNA-specific endonuclease DICER, which cleaves the targeting dsRNAs into 21�25-nucleotide fragments. The double-stranded siRNAs associate with the RNA-interference silencing ribonucleoprotein complex (RISC). The RISC contains adaptor proteins that bind to the siRNAs and unwind them (helicase activity) to generate single-stranded siRNAs. Another protein in the RISC, Argonaute 2, has RNase or SLICER activity. It cleaves the targeted RNAs (in this case the influenza virus RNA).
For targeted gene silencing, synthetic short double-stranded siRNAs are transfected into cells. A simplified schematic of siRNA-mediated degradation of influenza virus RNA is shown in [[Figure 1. Short-interfering RNA (siRNA)-mediated degradation of influenza polymerase (PA) RNA. To target specific RNAs for degradation, such as influenza PA, complementary synthetic short double-stranded siRNAs are transfected into cells. The double-stranded siRNAs are bound by RNA-interference silencing ribonucleoprotein complexes (RISCs). A helicase protein within the RISCs unwinds the two strands and generates single-stranded siRNAs. The single-stranded antisense siRNAs then bind to complementary influenza PA RNAs and guide SLICER (Argonaute 2) to specifically cleave the influenza PA RNAs at the site of siRNA binding.]].
The synthetic short double-stranded siRNAs do not require the DICER activity that is necessary for the processing of long dsRNAs. Long dsRNAs are found in virus-infected cells, but generally not in normal eukaryotic cells. The problem with using long dsRNAs is that they activate the interferon (IFN) system and the dsRNA-dependent IFN-inducible protein kinase (PKR). This leads to effects on cytokine signaling and transcription, the degradation of mRNA, the inhibition of translation and cell death. To avoid the adverse effects of long dsRNAs, viruses have evolved dsRNA binding proteins that can inhibit the effects of IFN and antiviral RNA interference. What makes short double-stranded siRNAs more useful for targeted gene silencing is that they induce the homologous sequence-dependent degradation of mRNAs without activating the IFN system. As with the in vivo impact of duplexed siRNAs against IAV observed by Ge et al. and Tompkins et al., the sequence specificity and virus specificity further control for potential IFN induction by dsRNAs and demonstrate that the antiviral effects are not due to IFN. siRNAs have been touted as a panacea for a wide range of applications. They have become standard in vitro laboratory tools to block specific gene activity, through the degradation of mRNAs, to inhibit virus replication. Recent reviews emphasize that the use of siRNAs holds great promise not only for combating viruses and other human pathogens but also allergies, tumors, pain and neurological diseases.